title: Evaluation of quantitative performance of sequential immobilized metal affinity chromatographic enrichment for phosphopeptides.
abstract: We evaluated a sequential elution protocol from immobilized metal affinity chromatography (SIMAC) employing gallium-based immobilized metal affinity chromatography (IMAC) in conjunction with titanium dioxide-based metal oxide affinity chromatography (MOAC). The quantitative performance of this SIMAC enrichment approach, assessed in terms of repeatability, dynamic range, and linearity, was evaluated using a mixture composed of tryptic peptides from caseins, bovine serum albumin, and phosphopeptide standards. Although our data demonstrate the overall consistent performance of the SIMAC approach under various loading conditions, the results also revealed that the method had limited repeatability and linearity for most phosphopeptides tested, and different phosphopeptides were found to have different linear ranges. These data suggest that, unless additional strategies are used, SIMAC should be regarded as a semiquantitative method when used in large-scale phosphoproteomics studies in complex backgrounds.
authors.affiliation: Department of Chemical and Biological Engineering, Colorado State University, Fort Collins, CO 80523, USA.
authors.forename: Zeyu
authors.initials: Z
authors.lastname: Sun
authors.forename: Karyn L
authors.initials: KL
authors.lastname: Hamilton
authors.forename: Kenneth F
authors.initials: KF
authors.lastname: Reardon
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002846:Chromatography, Affinity; D005708:Gallium / Q000737:chemistry*; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D012710:Serum Albumin, Bovine / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014025:Titanium / Q000737:chemistry*; D014357:Trypsin / Q000378:metabolism
chemical_list: D002364:Caseins; D010748:Phosphopeptides; C009495:titanium dioxide; D012710:Serum Albumin, Bovine; D005708:Gallium; D014025:Titanium; D014357:Trypsin
keywords: Affinity chromatography; IMAC; MOAC; Phosphopeptides; Proteomics; SIMAC